During the development of the human placenta, mononuclear cytotrophoblast (CTB) cells proliferate and fuse to form a syncytiotrophoblast (STB) phenotype. Although this differentiation process is critical for a successful pregnancy, the molecular mechanisms that regulate the process are poorly understood. Our laboratory, utilizing an in vitro model of human CTB cell differentiation, has shown that the transcription factor AP-21 is critical for the differentiation of villous CTB cells to a STB phenotype; and we have characterized some of the upstream regulators and downstream targets of AP-21. Our preliminary studies indicate that several extracellular factors that induce CTB cell differentiation stimulate AP-21 expression that the Wnt-beta-catenin- TCF pathway inhibits AP-21 expression during CTB cell differentiation and that AP-21 expression is abnormal in placentas from patients with severe preeclampsia. We now propose a comprehensive investigation into the mechanisms of which AP-21 regulates CTB cell to STB cell differentiation in the context of normal differentiation and in the context of pathologic conditions characterized by abnormal villous CTB differentiation, such as preeclampsia and IUGR. Specific Aim 1 tests the hypothesis that extracellular factors that induce villous CTB differentiation act, at least in part, by an AP-21-dependent mechanism. Aim 2 tests the hypothesis that there is a negative feedback loop between AP-21 and the Wnt-2-catenin pathway and that extracellular factors that induce CTB cell differentiation act at least in part by inhibiting Wnt-2-catenin-TCF signaling. Aim 3 examines the hypothesis that abnormalities in the AP-21 cascade are responsible, at least in part, for the defective villous CTB cell differentiation in preeclampsia and IUGR. The experiments will utilize an in vitro model of human CTB cell differentiation that closely mimics the in vivo differentiation process. The degree of differentiation will be monitored by cell morphology and by the expression of specific STB marker genes. The studies should provide new insights into the molecular mechanisms of placental differentiation and the pathogenesis of the defective placentation in preeclampsia and IUGR. These insights in turn may lead to development of new strategies to treat placental defects that result in fetal morbidity and mortality. PUBLIC HEALTH RELEVANCE: The proposed research examines the critical roles for the transcription factor AP-21 in the regulation of the differentiation of human villous cytotrophoblast cells to a syncytiotrophoblast cell phenotype. A comprehensive knowledge of the upstream regulation of AP-21 expression and the downstream targets of AP-21 action will provide important new information about the molecular pathophysiology of the placental abnormalities in preeclampsia, intrauterine growth retardation and other pathologic conditions of pregnancy.